1. Field of the Invention
This invention relates to a quantitative assay, applicable to clinical samples, for substrates which can undergo enzymatic hydrolysis to release long chain fatty acids. The invention is particularly, though not exclusively, directed to ester substrates such as the triglyceride substrates for lipase and phospholipid substrates for the phospholipases.
2. Description of the Prior Art
Esters such as the tri and diglycerides are ubiquitous and fundamental to every aspect of cell membrane function and energy transfer. The assay of these components is therefore of interest in many areas of clinical diagnosis. Thus, for example, assay of the triglyceride content of a clinical specimen can give some indication of recent dietary fat intake, and the ability of the liver to metabolise fats for energy utilisation. However presently available methods for assay of triglycerides, such as those available commercially as Kits from the Sigma Chemical Co. Ltd. (Poole, Dorset, UK) rely on the measurement of glycerol released by enzyme hydrolysis. Thus, in Sigma procedure no. 405, triglycerides are extracted into isopropanol and saponified with potassium hydroxide. Liberated glycerol is then converted to formaldehyde by periodate. By reacting with acetylacetone, the formaldehyde forms yellow diacetyldihydrolutidine, which is measured colorimetrically.
In Sigma procedures nos. 336, 337, 339 and 334 glycerol is released from triglyceride enzymatically using lipase, and glycerol is further reacted with ATP to form glycerol-1-phosphate. The four methods then differ only in the way by which the glycerol-1-phosphate is further reacted to produce a change in absorbance which can be measured spectrophotometrically. Such assays, when applied to clinical samples, suffer from the disadvantage that as glycerol itself is a product of cell metabolism, assay of the glycerol content of a blood specimen may not give an accurate picture of the circulating triglyceride levels in the subject (see Cole, Clin. Chem. 36/7, 1267-1268 (1990)). There is clearly a need for the development of alternative means for assaying triglycerides and other related substrates, such as phospholipids and cholesterol esters, that give accurate results at low concentrations or in small clinical blood specimens.